The correct secondary structures and the relationships of structural elements to the function of snRNAs in splicing have been appreciated for a number of years. Roles of snRNAs in recognition of each other and the premessenger RNA reactive sites that establish the structure of the active spliceosome have been demonstrated. A continuing mystery in the analysis of splicing is how the snRNA rearrangements are achieved and coordinated during assembly of the spliceosome, catalysis, and disassembly. A second major issue concerns how, at the biochemical level, the regulation of splicing impinges on the basic process of spliceosome assembly. A third and emerging set of questions concerns the role of splicing in genome function and evolution. Experiments addressing each of these major questions are proposed. By characterizing the functional interactions between U2 snRNA, the DExD/H ATPase Prp5p and Cus2p, the enforcer of ATP-dependence at the Prp5p step it will be possible to understand the mechanism of prespliceosome assembly. Critical to this process, the yeast SF3a (Prp9p, Prp11p, Prp21p), and SF3b (Cus1p, Hsh49p, Hsh155p, Rse1p) protein complexes are present near the catalytic core of the spliceosome. The hypothesis is that protein factors that interact with U2 snRNA play essential roles in the regulation, progression and fidelity of splicing reactions. A unique opportunity to study positive regulation of splicing is by exploring the functional interactions between Mer1p and the U1 snRNP during splicing activation. The hypothesis is that Mer1p accelerates a key step in spliceosome assembly or splicing. Finally a modified microarray technology capable of monitoring all introns in the yeast genome in parallel will be applied to questions concerning the global regulation of splicing. The results should reveal general features of presliceosome assembly in terms of the basal machinery at the key step of prespliceosome formation, and in instances of splicing regulation. Genome-wide views of splicing have never been developed, and the new technology will allow this important process to be understood in terms of regulatory networks. These studies will be important foundations for investigations into systems with more complex splicing, such as human cells.